DNA microarray hybridization technology has been applied to various kinds of studies. (Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science, 1995, 270, 467-70). DNA microarray hybridization has been an important and popular tool for broad applications in biomedical research. It is a valuable method for detecting up- or down-regulation of gene expression. In addition to gene expression, other major applications of DNA microarrays include pathogen detection, genotyping, resequencing, drug discovery (Lindsay, M. A. Nat. Rev. Drug Discov. 2003, 2, 831-8), pharmacogenomic research [Koch, W. H. Nat. Rev. Drug Discov. 2004, 3, 749-61], cancer diagnostics [Cheang, M. C.; van de Rijn, M.; Nielsen, T. O. Annu. Rev. Pathol, 2007, 3, 67-97] and protein-DNA interactions [Stoughton, R. B. Annu. Rev. Biochem. 2005, 74, 53-82, Heller, M. J. Annu. Rev. Biomed. Eng. 2002, 4, 129-53; Gunderson, K. L.; Steemers, F. J.; Lee, G.; Mendoza, L. G.; Chee, M. S. Nat. Genet. 2005, 37, 549-54; Gunderson, K. L.; Steemers, F. J.; Ren, H.; Ng, P.; Zhou, L.; Tsan, C.; Chang, W.; Bullis, D.; Musmacker, J.; King, C.; Lebruska, L. L.; Barker, D.; Oliphant, A.; Kuhn, K. M.; Shen, R. Methods Enzymol. 2006, 410, 359-76].
Evaluating the activity of various enzymes is essential for understanding of cellular mechanisms, such as intracellular signaling, division and growth processes. Generally, methods of measuring enzymatic activity are performed through antigen-antibody reactions in solution, such as the enzyme-linked immunosorbent assay (ELISA), but chip-based methods have recently been introduced for high-throughput analysis (MacBeath, G. and Schreiber, S. L. 2000, Science, 289, 1760-1763). In particular, a peptide chip has been popularly employed as a screening method of enzyme activity, due to the functional stability, facile synthesis of the substrate, and reproducible binding affinity (Reimer, U., Reineke, U. and Schneider-Mergener, J. Cur. Opi. Biotech. 2002, 13, 315-320). However, the peptide chip also depends mainly on fluorescence or radioisotope-labeled methods in the final analysis step, which are still time-consuming and labor-intensive. In addition, mass spectrometry allows for significant applicability for enzyme assay as a non-labeling method when applied to the peptide chip. Widely used mass spectrometry includes electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Technology of applying mass spectrometry to the chip surface typically involves surface-enhanced laser desorption/ionization (SELDI) spectrometry, and the Mrksich's group carried out mass spectrometry for measuring enzymatic activity and screening an inhibitor on the peptide chip using MALDI (Su, J. and Mrksich, M. Angew, Chem. 2002, 114, 4909-4912; D.-H., Tang, W-J. and Mrksich, M. 2004, Nanobiotech. 22, 717-723). Recently, gold nanoparticles (AuNPs) have been applied in various bioengineering fields. Particularly, in the field of mass spectrometry, there has recently been an example in which AuNPs were used as effective matrixes in MALDI (McLean, J. A., Stumpo, K. A. and Russel, D. H. 2005, J. Am. Chem. Soc. 127, 5304-5305). However, this example comprises mixing the gold nanoparticles with a target analyte in solution and then performing measurements on the surface and is not an example in which AuNPs are applied directly on the chip surface.
DNA microarray technology is a powerful tool for comparing levels of expression of large numbers of genes Oligonucleotide or complementary DNA (cDNA) probes are immobilized on solid phases such as modified glass or membrane surface by different technologies [R. B. Stoughton, Ann. Rev. Biochem. 74 (2005) 53-82]. An essential requirement for microarray analyses is that the probe spots be discreet and readily distinguishable from each other. Without this, no valid conclusions can be drawn. As a consequence, analysis of replicate array probes of the same sample is highly preferred in order to draw definitive conclusions about changes in gene expression.
The quality of arrays is critically important due to a large number of genes to be probed and detected on the microarray hybridization chip. DNA probes are first immobilized on the solid phase such as modified glass or membrane surface [Anthony, R. M.; Brown, T. J.; French, G. L. J. Clin. Microbiol. 2000, 38, 781-8]. DNA probes on a chip can be prepared either by in-situ synthesis method or spotting DNA segments onto the surface. DNA probes can be immobilized onto solid surface by different technologies such as Pen tip deposition [Yang, A. X.; Mejido, J.; Bhattacharya, B.; Petersen, D.; Han, J.; Kawasaki, E. S.; Puri, R. K. Mol Biotechnol. 2006, 34, 303-15], ink-jet deposition [Okamoto, T.; Suzuki, T.; Yamamoto, N. Nat. Biotechnol. 2000, 18, 438-41], photolithographic mask [Beier, M.; Hoheisel, J. D. Nucleic Acids Res. 2000, 28, e11], and bead array [Steemers, F. J.; Ferguson, J. A.; Walt, D. R. Nat. Biotechnol. 2000, 18, 91-4].
Microarray signals are detected by many technologies. Fluorescent labeling and detection is the most popular technique used to identify hybridization signals because it is sensitive and much easier and safer to handle than radioactive labeling methods [Parrish, M. L.; Wei, N.; Duenwald, S.; Tokiwa, G. Y.; Wang, Y.; Holder, D.; Dai, H.; Zhang, X.; Wright, C.; Hodor, P.; Cavet, G.; Phillips, R. L.; Sun, B. I.; Fare, T. L. J. Neurosci. Methods, 2004, 132, 57-68]. Sensitive fluorescence detection commonly uses a laser and a confocal microscope, e.g., DNA microarray detector made by Affymetrix Inc., which are typically very expensive and need a trained technician to operate.
Other technologies to detect microarray hybridization are microelectronic arrays [Westin, L.; Xu, X.; Miller, C.; Wang, L.; Edman, C. F.; Nerenberg, M. Nat. Biotechnol. 2000, 18, 199-204] and quantum dot methods. Different colors of quantum dots attached to oligonucleotides to hybridize with targets and the signals are detected at specific wavelengths [Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631-5; Liang, R. Q.; Li, W.; Li, Y.; Tan, C. Y.; Li, J. X.; Jin, Y. X.; Ruan, K. C. Nucleic Acids Res. 2005, 33, e17]. The accuracy is high, but the number of colors available to do hybridization is limited.
Nanoparticles have been recently introduced for detecting DNA microarray hybridization. DNA probes are synthesized on gold nanoparticles and hybridized with DNA on glass surface. The sensitivity of the gold labeling method is almost equal to that of fluorescent labeling [Cao, Y. C.; Jin, R.; Mirkin, C. A. Science, 2002, 289, 1757-60; T. A. Taton, C. A. Mirkin, R. L. Letsinger, Science 289 (2000) 1757-1760]. Silver enhancement is usually pursued to amplify the signal. However, skilful handling to prevent overstaining by silver is essential.
Most of microarray quality control studies to date have focused on post-hybridization data quality control rather than on probe spot quality measurements before hybridization. For example, the Microarray Quality Control (MAQC) project of the U.S. FDA is a platform that has been established to help microarray researchers compare inter- or intraplatform microarray results [MAQC Consortium, Shi, L. et al. Nat. Biotechnol. 2006, 24, 1151-61]. Bylesjö et al. assessed the cDNA microarray spot quality following hybridization with target genes labeled with Cy5 and Cy3 dyes [Bylesjö, M.; Eriksson, D.; Sjödin, A.; Sjöström, M.; Jansson, S.; Antti, H.; Trygg, J. BMC Bioinformatics, 2005, 6, 250-9]. Both methods assess quality after hybridization. Array quality could be used to check the general quality of hybridization, but the studies assume that the qualities of all chips were more or less the same. However, this is a risky assumption. The characterization of microarray spots for each chip is critical before the hybridization step.
Several microarray quality control methods for spots have been used. SYTO® 61 and SYBR® Green II dye staining techniques are array quality control methods before hybridization [Yue, H.; Eastman, P. S.; Wang, B. B.; Minor, J.; Doctolero, M. H.; Nuttall, R. L.; Stack, R.; Becker, J. W.; Montgomery, J. R.; Vainer, M.; Johnston, R. Nucleic Acids Res. 2001, 29, e41; Battaglia, C.; Salani, G.; Consolandi, C.; Bernardi, L. R.; De Bellis, G. BioTechniques, 2000, 29, 78-81]. These approaches use non-destructive DNA-binding fluorescent dye to stain DNA probes. Only a few slides in each batch are usually tested by these methods because the tested arrays are not available for subsequent hybridization [Shearstone, J. R.; Allaire, N. E.; Getman, M. E.; Perrin, S. BioTechniques, 2002, 32, 1051-7]. Wang et al. used TDAV (third dye array visualization) technology to quantitatively evaluate and control every array. [Wang, X.; Jia, S.; Meyer, L.; Xiang, B.; Chen, L. Y.; Jiang, N.; Moreno, C.; Jacob, H. J.; Ghosh, S.; Hessner, M. J. BMC Bioinformatics, 2006, 7, 378-87]. The fluorescein isothiocyanate (FITC) labeled DNA probes or oligonucleotides are printed on the array slides and makes prehybridization assessment of array quality possible. [Hessner, M. J.; Singh, V. K.; Wang, X.; Khan, S.; Tschannen, M. R.; Zahrt, T. C. BMC Genomics, 2004, 5, 12-22; Hessner, M. J.; Wang, X.; Hulse, K.; Meyer, L.; Wu, Y.; Nye, S.; Guo, S. W.; Ghosh, S. Nucleic Acids Res. 2003, 31, e14]. The advantage of the method is to eliminate substandard arrays before experiments. Probe retention rate can also be monitored, but to label DNA probes with fluorescence dyes is expensive and tedious. The detection also needs expensive confocal laser scanners to avoid overlap of the emission wavelengths of fluorochromes used in sample detection such as Cy3 or Cy5 [Yang, A. X.; Mejido, J.; Bhattacharya, B.; Petersen, D.; Han, J.; Kawasaki, E. S.; Puri, R. K. Mol. Biotechnol. 2006, 34, 303-15]. An electrochemical immunoassay has been developed using a colloidal gold label that, after oxidative gold metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltametry (ASV) at a single-use carbon-based screen-printed electrode (SPE). [Dequaire, M.; Degrand, C.; Limoges, B. Anal. Chem. 2000, 72, 5521-5528.]
A need exists in the art to address the aforementioned deficiencies and inadequacies, especially in connection with development of methods for characterization of chips prior to DNA microarray hybridization. As of this invention, there were no chips that allow both quality analysis and hybridization using the same chip. It is risky to draw conclusions from results of different chips if there is no knowledge of the quality of the chips before hybridization, even if they are from the same batch.
Due to the high cost and labor involved in fabricating microarrays, there also is a need for methods that allow multiple rounds of use of a microarray chip. Typically, hybridization reactions on a microarray surface are detected by staining. There exists a need for methods that allow destaining of a hybridized chip in a manner that render the destained chips suitable for subsequent rounds of hybridization reactions.